Ataxia-Telangiectasia (A-T) is caused by mutations in the ATM gene, which encodes the Ataxia-Telangiectasia Mutated kinase (ATM). Many symptoms of A-T, including neurodegeneration and cancer, are likely exacerbated by chronic oxidative stress due to increased ROS. Mitochondria add significantly to cellular ROS when electrons are prematurely transferred to oxygen during oxidative phosphorylation. The Sponsor's lab has linked mitochondrial dysfunction to increased ROS production in A-T patient cells, leading to the overall premise of this proposal that increased mitochondrial ROS (mROS) contribute to A-T pathology. Specifically, this proposal will test the hypotheses that mROS 1) are sensed by ATM to regulate mammalian target of rapamycin complex 1 (mTORC1)-mediated pro-growth and stress-response signaling and 2) contribute to increased activity of Akt, both of which likely contribute to A-T pathology. Independent of its role in DNA damage signaling, ATM is activated by ROS and inhibits mTORC1 via activation of AMP-activated protein kinase (AMPK). Since mTORC1 signaling stimulates growth and inhibits stress resistance and autophagy pathways, the hypothesis of Aim 1 is that ATM normally senses mROS in order to inhibit mTORC1 to repress cell growth and increase stress resistance capacity. Experiments will be carried out using mouse embryonic fibroblasts (MEFs) prepared from ATMflox/flox mice in which the ATM gene will be knocked out using a lentiviral- Cre system and acute changes in AMPK and mTORC1 signaling will be assessed by western blot of activated kinases and their downstream targets. The contribution of mROS will be determined by monitoring mTORC1 activation in ATMflox/flox MEFs that do or do not overexpress mitochondrial antioxidant enzymes (mCAT and MnSOD). Finally, to determine if ATM senses mROS and/or mitochondrial dysfunction, ATM activation will be measured in response to pharmacological agents that increase mROS, inhibit respiration, or both. Akt is a kinase involved in cell growth, proliferation survival, and metabolism pathways. Hyperactive Akt is observed in thymocytes in ATM-/- mice and in multiple tissues in ATM-/- mice. Akt is activated by PI3K signaling, which is negatively regulated by the tumor suppressor PTEN. Another potential mechanism of Akt hyperactivation is its inability to be polyubiquitinated and degraded. In Aim 2, the mechanism of Akt activation in ATM-/- MEFs will be determined by monitoring for changes in PTEN activity and/or in the synthesis and stability of Akt. Finally, since hyperactive Akt may upregulate mTORC1 through inhibition of TSC2, pharmacological and gene knockdown inhibition of Akt and analysis of mTORC1 signaling parameters will be carried out as described in Aim 1 to determine if this is the case in ATM-/- MEFs. The long-term goal of this project is to better understand the role of mROS in ATM and mTORC1 signaling, which may allow development of new therapeutic strategies for A-T.